Microbial Viability Assay Kit-WST

Protocol tips

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	Add 10 μL colouring reagent (comprising nine parts WST solution and one part electron mediator reagent for Gram-negative bacteria, or nine parts WST solution and one part eight-fold diluted electron mediator reagent for Gram-positive bacteria). Incubate the plate in the incubator for 2–3 h at 35 °C

Protocol tips
Add 10 μL colouring reagent (comprising nine parts WST solution and one part electron mediator reagent for Gram-negative bacteria, or nine parts WST solution and one part eight-fold diluted electron mediator reagent for Gram-positive bacteria). Incubate the plate in the incubator for 2–3 h at 35 °C

Publication protocol

The Microbial Viability Assay Kit-WST, purchased from Dojindo Laboratories (Kumamoto, Japan), offers a method of microbial metabolism detection by colorimetry. According to the manufacturer's technical manual, WST-8, employed as a colorimetric indicator, is directly proportional to the number of living microorganisms.
Since the species and the metabolic activity have effects on the sensitivity (O.D. value), previous work has been done to optimize the number of cells and colouring reaction time for each test. After cultivation overnight, a bacterial suspension of 107 CFU mL−1 using McFarland standards was prepared, further diluted with sterile saline to provide various densities, and then inoculated into a 96-well plate. After incubation for 6 h at 35 °C, 10 μL of the colouring reagent was added to each well, followed by another 2–3 h incubation. The O.D. value was measured at 450 nm with a microplate reader (Bio-Rad Laboratories, USA), and then the cell density corresponding to the O.D. value within a range of 2–4 was selected.

Bacteria were incubated at 37 °C in broth at 150 rpm overnight and then diluted to the proper density according to McFarland standards. After 90 μL MH broth was added to each well of columns 1 to 10 of the 96-well microplate, Ga(NO3)3 solution that had been prepared was added to the first well, followed by a serial two-fold dilution from columns 1 to 10. After that, another 90 μL MH broth was added to each well of columns 1 to 10, making the final volume 180 μL and dilutions of 1024 to 2 μg mL−1. Columns 11 and 12 were the positive and negative controls, respectively. A microbial suspension (10 μL) was inoculated into each well of columns 1 to 11, and the microplate was incubated for 6 h at 35 °C. Then, 10 μL colouring reagent (comprising nine parts WST solution and one part electron mediator reagent for Gram-negative bacteria, or nine parts WST solution and one part eight-fold diluted electron mediator reagent for Gram-positive bacteria) was added to each well of the microplate, with another incubation for 2–3 h at 35 °C. Finally, the microplate was read at 450 nm with a microplate reader (Bio-Rad Laboratories, USA), and the negative control was read as the blank value. The MIC value was defined as the lowest concentration of gallium nitrate solution where the absorbance change was no more than 0.05 compared to the blank value.30–32 The results are reported as the mean of the experiment that was conducted in triplicate.

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Manufacturer protocol

Download the product protocol from Dojindo for Microbial Viability Assay Kit-WST below.

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